Pharmaceutical composition and dietary supplement containing docosarexaenoic acid obtained from dinoflagellates

ABSTRACT

A pharmaceutical composition and a dietary supplement comprising a single cell oil containing at least 20% docosahexaenoic acid (DHA) in triglyceride form are presented. The single cell oil is extracted from biomass produced by cultivating heterotrophic microalgae of the class Dinophyceae in an aerobic fermentor with a nutrient solution having a limited nitrogen source.

This application is a division of application Ser. No. 07/916,874, filedAug. 13, 1992, now U.S. Pat. No. 5,397,591, which was based onPCT/US91/00733/ filed Feb. 4, 1991 and a continuation-in-part of U.S.Ser. No. 07/479,135, filed Feb. 13, 1990, now U.S. Pat. No. 5,407,957.

BACKGROUND OF THE INVENTION

This invention relates to edible, single-cell oil containingdocosahexaenoic acid (DHA). The invention also relates to methods ofproducing such oil containing DHA in commercially viable yields and toproducts containing the oil.

DHA is an omega-3-fatty acid and is the most abundant long chainpolyunsaturated fatty acid (PUFA) in the grey matter of the brain.Omega-3-fatty acids in general are. known to be beneficial in reducingthe incidence of coronary heart disease [Lands, Fish and Human Health(1986) Academic Press]. However, the metabolism of omega-3-fatty acidsis not well understood. Thus, precise clinical dosages and efficacyremain unknown.

Cold water marine fish are a known source of omega-3-fatty acids,including DHA. U.S. Pat. No. 4,670,285 discloses the use of fish oilfrom fish such as menhaden and herring as a source of C₂₂ omega-3-fattyacids. Indeed, fish oils are the primary commercial source ofomega-3-fatty acids. Often, however, fish oils are unusable for humanconsumption because of contamination with environmental pollutants suchas PCB's.

There also are problems associated with the recovery of fish oilscontaining DHA for food uses. Such oils often have a fishy odor andunpleasant tastes associated with the oxidation products of the fattyacids. These tastes and toxicities of peroxides render the oilsunsatisfactory for use in edible compositions such as baby food andinfant formulas.

Marine microorganisms also are known to contain DHA. In particular,various species of dinoflagellates are known to contain DHA. Harringtonet al., "The Polyunsaturated Fatty Acids of Marine Dinoflagellates" J.Protozoal, 17:213-219 (1970), characterize the fatty acid content ofeight photosynthetic and one heterotrophic marine dinoflagellates, andconclude that the dinoflagellates are a primary producer group ofdocosahexaenoic acid and contribute substantial amounts of that compoundto the marine food chain.

Successful cultivation of dinoflagellates to produce an edible oilcontaining DHA has not been achieved. Dinoflagellates in general arevery slow growing and are shear sensitive. Guillard et al.,Dinoflagellates, (1984) Academic Press. The prior art discloses thateven a small amount of agitation in the culturing vessel reduces growthof the cultures. However, such agitation would be necessary to achieveadequate oxygenation in order to maximize growth for commercialproduction.

DHA is thought to be essential for the proper brain and visiondevelopment of infants because, as noted above, it is the most abundantlong chain PUFA in the brain and retina. Although a metabolic pathwayexists in mammals for the biosynthesis of DHA from dietary linolenicacid, this pathway is bioenergetically unfavorable [Crawford, P. AOCS.Short Course in Polyunsaturated Fatty Acids and Eicosanoids, pp. 270-295(1987)] and mammals, like fish, are thought to obtain most of their DHAfrom dietary sources. In the case of infants, the most likely sourcewould be human milk. Indeed, DHA is the most abundant C20 omega-3 PUFAin human milk. Generally, however, DHA is absent from infant formulas.U.S. Pat. No. 4,670,285 does disclose an infant formula containingomega-3-fatty acids. However, the acids utilized therein are obtainedfrom egg or fish (Talapia) oil and have associated therewith theunpleasant characteristics previously described. Furthermore, fish oilsgenerally contain another omega-3-fatty acid, eicosapentaenoic acid(EPA), an undesirable component in infant formulas because of itsprolonged anticoagulant effects and its depression of arachidonic levelsin infants. This has been correlated with reduced rates of infant weightgain (Carleson et al. INFORM 1:306.) Indeed, EPA levels are very low inhuman milk (less than one-forth that of DHA).

Accordingly, it is an object of the present invention to provide asingle-cell edible oil containing DHA. Preferably this oil will have nosignificant quantities of other polyunsaturated fatty acids (PUFA's),i.e. greater than about 2% of the total fatty acid content. In general,it is an object of the present invention to produce single-cell oil incommercially viable yields. The oil, characterized herein as a"designer" oil, after extraction can be used in infant formulas, babyfoods, dietary supplements and pharmaceuticals.

In addition, it would be desirable to acquire further knowledge of themetabolic pathway of omega-3-fatty acids. Isotopically labeled DHA wouldbe of great utility in this regard. However, to date, no method has beenknown to produce abundant quantities of isotopically labeled DHA. Thus,it also is an object of the present invention to provide isotopicallylabeled DHA in sufficient quantities to undertake such research.

SUMMARY OF THE INVENTION

The present invention relates to the cultivation of microorganisms,notably dinoflagellates, in a fermentor, induction of thosemicroorganisms to produce significant quantities of-single cell oilcontaining a high proportion of DHA and recovery of that oil. As usedherein, "single cell oil" refers to a lipid product of a unicellularorganism. The present invention also includes mutant organisms capableof producing enhanced quantities of single-cell oil containing at leastabout 20% by weight DHA and includes single cell oil containing DHA.

The present invention provides an economical method of obtainingenhanced levels of edible oils containing DHA. Additionally, the methodpermits the commercial cultivation of dinoflagellates in elevated celldensities.

Edible oils produced by the method of this invention lack unpleasanttastes and fishy odors and also are free of environmental contaminantsoften found in DHA-containing oils from conventional sources.Accordingly, the present invention further includes food productscontaining the oil of this invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1, 2 and 3 are graphic illustrations of C. cohnii biomassaccumulation over time with the addition of various nutrients.

DETAILED DESCRIPTION OF THE BEST MODE OF PRACTICING THE INVENTION

In accordance with the present invention, microorganisms capable ofproducing a single cell oil containing DHA are cultivated in a fermentorin a nutrient solution capable of supporting the growth of suchorganisms. Preferably the single cell oil will contain at least about20% by weight DHA.

Any microorganisms capable of producing a single-cell edible oilcontaining DHA can be used in the present invention. For example,photosynthetic diatoms can be used. Preferred microorganisms are marinedinoflagellates, including Crypthecodinium sp. Especially preferred, isCrypthecodinium cohnii, an obligate heterotroph requiring a reducedcarbon source for growth. C. cohnii is preferred because it contains afatty acid profile in which DHA is the only PUFA present in sufficientquantities (greater than about 1% of the total amount of PUFAs). Samplesof this organism, designated MK8840, have been deposited with theAmerican Type Culture Collection at Rockville, Md., and assignedaccession number 40750. As used herein, microorganism, or any specifictype of microorganism, includes wild strains, mutants or recombinanttypes. Any microorganism which produces enhanced levels of oilcontaining DHA is considered to be within the scope of this invention.One of the features of the present invention is its recognition of theedible oil-producing capability of microorganisms such asdinoflagellates and the attendant solution to the problem of maintaininga reliable, economic source of such oils. Accordingly, wild-type andrecombinant microorganisms designed to produce single cell oilcontaining DHA are an aspect of this invention. Such recombinantorganisms would include those designed to produce greater quantities ofDHA in the single cell oil, greater quantities of total oil, or both, ascompared to the quantities produced by the same wild type microorganism,when provided with the same substrates. Also included would bemicroorganisms designed to efficiently use more cost-effectivesubstrates while producing the same amount of single cell oil containingDHA as the comparable wild-type microorganism.

In general, those of skill in the art would not consider C. cohnii asuitable organism for cultivation in a fermentor. Previous workers havecommented on the extremely complex mixture of nutrients required tosuccessfully cultivate C. cohnii. Gold et al. Protozoal, 13:255-257(1966); Guillard, et al. in "Dinoflagellates", Academic Press (1984);Henderson, et al., Phytochemistry 27:1679-1683 (1988). In contrast, thepresent invention achieves the cultivation of DHA-producingmicroorganisms in a simple medium containing glucose and yeast extract.Use of these components in a solution such as seawater provideseconomically significant growth rates and cell densities. For example,during the course of a 3-5 day fermentation, C. cohnii cell densities ofat least 10 grams of biomass per liter of solution, and typically from20 to about 40 grams per liter, can be attained. Such densities have notheretofore been attainable.

Although cultivation can occur in any suitable fermentor, preferably theorganism is grown either in a stirred tank fermentor (STF) or in an airlift fermentor (ALF), both types known to those of skill in the art.When a STF is selected, agitation is provided using either Rushton-typehigh efficiency turbines or pitched-blade or marine impellets. Agitationand sparging renew the supply of oxygen to the microorganisms. The rateof agitation normally is increased as the biomass increases, due to theincreased demand for oxygen. It is desirable to keep the tip speed atnot greater than about 500 cm/sec, preferably not greater than about 300cm/sec. Selection of strains of microorganisms which are capable ofwithstanding greater tip speeds without undergoing shear is within thepurview of those of skill in the art. The use of such strains isexpressly included in this invention.

As noted above, seawater is an acceptable medium for the nutrientsolution. The seawater can be either natural, filtered or an artificialmix, each of which can be diluted to reduced salinities, such as 1/2 to1/4 normal strength, with tap water or concentrated to 2 times normalstrength. A preferred example is Instant Ocean® (IO) brand artificialseawater. Although C. cohnii is a marine microorganism, some growth hasbeen observed in zero salinity. The use of variants which grow well inreduced salinities is specifically encompassed by this invention.Micronutrients can be added and may be required at low salinities.However, such micronutrients are known to those of skill in the art andgenerally are present in seawater or tap water. If the organism selectedis heterotrophic, such as C. cohnii, then a carbon source is added.

Preferably, after addition of the seawater medium to the fermentor, thefermentor containing the medium is sterilized and cooled prior to addingthe nutrients and a seeding population of microorganism. (Although it isacceptable to sterilize the nutrients together with the seawater,sterilization in this manner can result in a loss of available glucose.)The nutrients and microorganism can be added simultaneously orsequentially.

An effective seed concentration can be determined by those of skill inthe art. When a STF is used, the addition of a population of from about0.05 to 1.0 grams of dry weight equivalent per liter at the beginning ofthe fermentation is preferred. This is about 10⁶ cells per ml. Thus, fora 30 liter fermentor, 1-3 liters of seeding media, containing viablecells at a density of 20 g dry weight per liter would be added.

Oxygen levels preferably are maintained at a D.O. of at least about 10%of air saturation level. Biosynthesis of DHA requires oxygen and,accordingly, higher yields of DHA require D.O. levels at from about 10%to 50% of air saturation levels. Agitation tip speeds of 150-200 cm/secin combination with an aeration rate of 1 VVM (volume of air/volume offermentor per minute) provides D.O. levels of from about 20% to about30% at biomass densities of about 25 g dry weight/liter of culture.Higher cell densities may require higher D.O. levels, which can beattained by increased aeration rates by O₂ sparging, or by increasingthe air pressure in the fermentor.

Acceptable carbon sources are known to those of skill in the art. Forexample, carbon can be provided to C. cohnii in the form of glucose.Other heterotrophs can use other reduced carbon sources, a matter easilydetermined by those of skill in the art, and autotrophs utilize carbondioxide. C. cohnii will also grow on other reduced, more complex, carbonsources. Typically, a fermentation is initiated with about 10-50 g/literglucose. More glucose is added during the fermentation as required.Alternatively, from about 50 to 150 g, preferably 50 to 100 gglucose/liter initially can be added, thereby minimizing the frequencyof future additions. The amount of carbon source provided to otherorganisms can readily be determined by those of skill in the art.

In addition to a reduced carbon source, a nitrogen source, such as yeastextract (YE), is provided to the medium. Commercially available yeastextract is acceptable. For example, DIFCO or MARCOR brand yeast extractcan be used. The yeast extract is an organic nitrogen source alsocontaining micronutrients. Other organic nitrogen sources easily can bedetermined by those of skill in the art. However, such compounds aregenerally more expensive than yeast extract. The use of variants capableof growing on urea or nitrates is within the scope of this invention.Typically, the fermentation is initiated with about 6-12 g YE/liter.More YE can be added as required. A typical fermentation run requiresfrom about 8 to 15 g YE/liter over the course of the run. Accordingly,that amount of YE can be added initially with a reduced need for furtheradditions. The precise amount can be determined by those of skill in theart. Generally, the ratio of glucose to YE is from about 2:1 to about15:1.

The cultivation can be carried out at any life-sustaining temperature.Generally C. cohnii will grow at temperatures ranging from about 15° C.to 34° C. Preferably the temperature is maintained at about 20°-30° C.Strains which grow at higher temperatures are preferred, because theywill have a faster doubling time, thereby reducing the fermentationtime. Appropriate temperature ranges for other microorganisms arereadily determined by those of skill in the art.

The cultivation can be carried out over a broad pH range, typically fromabout pH 5.0 to 9.0. Preferably, a pH range of from about 6.0 to about7.0 is used for the growth phase. A base, such as KOH or NaOH, is usedto adjust the media pH prior to inoculation. During the later stages ofthe fermentation, the culture medium tends to become alkaline. Ifdesired, inorganic acid pH controls can be used to correct alkalinityduring the growth phase.

Production of the single cell oil is induced in the dinoflagellates bythe imposition of a stationary phase (i.e., by nitrogen depletion or apH rise). YE deficiencies are caused by providing YE in a limitingamount such that the medium runs out of YE while available glucoseremains. The present invention recognizes that it is the carbon sourceto nitrogen source ratio which promotes the efficient production of thesingle cell oil. Using glucose and YE as exemplary, a preferred ratio ofcarbon source to nitrogen source is about 10-15 parts glucose to 1 partYE. Similar ratios for other carbon and nitrogen sources can becalculated by those of skill in the art.

After induction of oil production, the culture is grown for about 24additional hours. During this period of oleosynthesis, the single celloil containing DHA is being synthesized and visible oil droplets becomeapparent. Those of skill in the art can readily calculate the time offermentation required to achieve the expected amount of cell biomassbased upon the added amount of YE. When that time has passed, theculture is grown for an additional 24 hours and harvested. In general,the C. cohnii are cultivated for a time sufficient to produce singlecell oil, usually from about 60 to about 90 hours, although this time issubject to variation.

From about 15 to 30% of the resultant biomass, using wild-type C.cohnii, comprises extractable oil. Strain selection can increase thispercentage and such selection is within the scope of this invention.Preferably, the oil comprises greater than about 70% triglycerideshaving, in general, the following fatty acid composition.

15-20% myristic acid (C_(14:0))

20-25% palmitic acid (C_(16:0))

10-15% oleic acid (C_(18:0))

30-40% DHA (C_(22:6))

0-10% others

(Other oil components including polar lipids, such as phosphatidylcholine, also may be enriched in DNA.) The crude oil is characterized bya yellow-orange color and is liquid at room temperature. Desirably, theoil contains at least about 20% DHA by weight and most preferably atleast about 35% DHA by weight.

The organisms are harvested by conventional means, known to those ofskill in the art, such as centrifugation, flocculation or filtration,and can be processed immediately or dried for future processing. Ineither event, the oil can be extracted readily with an effective amountof solvent. Suitable solvents can be determined by those of skill in theart. However, preferred solvents include pure hexane and supercriticalfluids, such as supercritical CO₂.

Extraction techniques using supercritical fluids are known to those ofskill in the art and described in McHugh et al., Supercritical FluidExtraction, Butterworth, 1986. If the extraction solvent is hexane, asuitable ratio of hexane to dry biomass is about 4 liters of hexane perkilogram of dry biomass. The hexane preferably is mixed with the biomassin a stirred reaction vessel at a temperature of about 20°-50° C. forabout 2 hours. After mixing, the biomass is filtered and separated fromthe hexane containing the oil. Alternatively, a wet biomass paste(30-35% solids) can be extracted directly with more polar solvents, suchas ethanol, isopropanol or hexane/isopropanol mixtures. The residualbiomass, i.e. the single cell edible oil extracted biomass of themicroorganisms, such as C. cohnii, can be used as an animal feed,containing as it does about 35-40% protein, 8-10% ash and 45-50%carbohydrates. Because of this high protein content and the elevatedlevels of DHA, the whole biomass paste can be used for aquaculture(e.g., shrimp, oysters, fish) feed.

The solvent then is removed from the oil by distillation techniquesknown to those of skill in the art. Conventional oilseed processingequipment is suitable to perform the filtering, separation anddistillation. Additional processing steps, known to those of skill inthe art, can be performed if required or desirable for a particularapplication. These steps also will be similar to those involved inconventional vegetable oil processing and allow the separation ofDHA-enriched polar lipid fractions.

Isotopically labeled single cell oils, including labeled DHA, can beeasily obtained in sufficient quantities to permit research into themetabolic pathways of DHA by the method of this invention. When ¹³C-glucose or ¹⁴ C-glucose is provided as the reduced carbon substrate,labeled DHA results.

The present invention also includes food products, such as infantformulas and baby foods, as well as dietary supplements, which containthe single-cell oil containing DHA of the present invention. While thoseof skill in the art have recognized that infant formulas containing DHAare desirable, the prior art infant formulas contained DHA from fishoil, with its attendant unpleasant tastes and organolepticcharacteristics. Furthermore, fish oil supplementation of infant formulaincludes the addition of eicosapentaenoic acid (EPA), an omega-3-fattyacid known to possess anticoagulant activity and possibly responsiblefor reduction of arachidonic acid biosynthesis. Such an activity is notdesirable in infant formula or baby food and the single cell oildescribed herein contains no significant quantity of EPA. Food products,such as infant formula, containing the single cell oil of the presentinvention do not have the unpleasant organoleptic characteristics offish oil. The food products thus are more readily accepted by infantsand adults alike. Preferably the infant formula of the present inventioncontains about 0.05% by weight of single cell oil containing DHA. Thebaby food of the present invention, having a more solid constitution,preferably contains about 0.5% by weight of single cell oil containingDHA. In both instances, most preferably, the oil contains at least about35% DHA.

The present invention includes pharmaceutical products including singlecell oil containing DHA. Preferably the products contain at least about35% DHA. Exemplary of such pharmaceutical products is one suitable foruse in providing total parenteral nutrition (TPN) to infants or adults.Additionally, dietary supplements containing the single cell oil areencompassed. Preferably, such supplements are in the form of gelatincapsules encapsulating said oil and may be appropriate for pregnantwomen or breast feeding mothers. This especially may be true for suchwomen who are vegetarians and do not get sufficient amounts of DHA intheir diets.

The present invention also includes single cell oil containing DHA.Preferably the single cell oil contains at least about 20% by weightDHA. Most preferably the oil contains at least about 35% by weight DHA.

The present invention having been generally described, reference is hadto the following non-limiting specific examples.

EXAMPLE 1

Into a 30-liter working volume STF was loaded a medium of one halfstrength artificial seawater. Six liters of IO were combined with 18liters of tap water. The fermentor containing the medium was sterilizedand cooled to 28° C. Four hundred ml of concentrated YE (455 g/l), 900ml of glucose syrup (400 g/l) and one liter of inoculum from a seedfermentor containing about 2×10⁷ cells/ml or a biomass of 20 g/liter(yielding a final concentration of about 7×10⁶ cells/ml or a biomass ofabout 700 mg/liter), were added to the medium. Agitation was set at 120cm/sec tip speed and aeration was set at 1 VVM (30 liters per minute).Additional glucose syrup (900 ml) was added after 30 hours and another4.2 liters over the next 42 hours. Thus 6 liters of glucose syrup wereadded in total. Concentrated YE solution (400 ml) was added at hour 6and another 1.2 liters were added over the next 48 hours until a totalof 2.0 liters had been added. To maintain the D.O. at greater than 20%,at 24 hours the agitation tip speed was increased to 150 cm/sec and at48 hours to 160 cm/sec. At 72 hours, the tip speed was increased to 200cm/sec and the culture was permitted to grow for an additional timesufficient to convert the final charge of glucose into cellular oil. Theculturing conditions are depicted graphically in FIG. 1. The culture wasthen harvested by centrifugation with the cell pellet retained. Theharvested pellet of cells was frozen and dried (lyophilized) to about a4% moisture content. Hexane (2.8 liters) was added to the dried biomassand stirred in a glass kettle for 1.5 hours at 50° C. A rotaryevaporator was used to remove the hexane, producing about 175 g of crudeDHA-containing oil.

EXAMPLE 2

Into a 350-liter working volume STF was loaded a medium of one halfstrength artificial seawater made by combining 4.3 kg. of I.O.® with 230liters of tap water. The fermenter containing the medium was sterilizedand cooled to 28° C. 6.8 liters of concentrated YE (400 g/l), 12.5liters of glucose syrup (400 g/l) and 0.30 liters of C. cohnii inoculumfrom a seed fermenter (10⁶ cells/ml or a biomass density of about 1.3g/liter) were added to the medium. Agitation was set at 73 cm/sec tipspeed and aeration was set at 1 VVM (280 liters per minute). Additionalglucose syrup (12 liters) was added after about 44 hours and another 43liters over the next 32 hours. Thus, 67.5 liters of glucose syrup wereadded in total. The glucose additions and the cell growth are depictedgraphically in FIG. 2.

To maintain the D.O. at greater than 20%, at 44 hours the agitation tipspeed was increased to 175 cm/sec and at 55 hours to 225 cm/sec. At 76hours, the tip speed was decreased to 150 cm/sec and the culture waspermitted to grow for an additional time sufficient to convert the finalcharge of glucose into cellular oil. The culture then was harvested. Theharvested cells were dried to about a 4% moisture content. Hexane wasadded to the dried biomass and stirred in a glass kettle for 2 hours at25° C. A rotary evaporator was used to remove the hexane, producingabout 700 g of crude DHA-containing oil.

EXAMPLE 3

Into a 30-liter working volume STF was loaded a medium of full strengthartificial seawater made by combining 565 g of I.O.® with 15 liters oftap water. The fermenter containing the medium was sterilized and cooledto 28° C. Four hundred ml of concentrated YE (400 g/l), 1.9 liters ofglucose syrup (400 g/l) and 1 liter of C. cohnii inoculum from a seedfermenter (10⁶ cells/ml or a biomass of about 2.0 g/liter) were added tothe medium. Agitation was set at 80 cm/sec tip speed and aeration wasset at 1 VVM (20 liters per minute). Additional glucose syrup (1.5 l)was added after 94 hours and another 1.1 liters at 116 hours. Thus 4.5liters of glucose syrup were added in total. To maintain the D.O. atgreater than 20%, at 52 hours the agitation tip speed was increased to160 cm/sec. At 66 hours, stationary phase was induced and in order toaccomplish this, the pH was spiked with 4N KOH to 7.0 and the agitationtip speed was not further increased for the duration of the run. Asshown in FIG. 3, the culture was permitted to grow for an additionaltime sufficient to convert the final charge of glucose into cellularoil. The culture then was harvested. The harvested cells were dried toabout a 4% moisture content. Hexane was added to the dried biomass andstirred in a glass kettle for 1.5 hours-at 50° C. A rotary evaporatorwas used to remove the hexane, producing about 65 g of crudeDHA-containing oil.

We claim:
 1. A pharmaceutical composition comprising a single cell oilcontaining at least 20% docosahexaenoic acid (DHA) in the triglycerideform, produced by a process comprising:a) cultivating a heterotrophicmicroalgae of the class Dinophyceae capable of producing a single celloil containing at least 20% DHA in the triglyceride form in an aerobicfermentor containing a nutrient solution having a limiting nitrogensource, said cultivation being continued to achieve a cell density of atleast 10 grams of biomass per liter of nutrient solution, theconcentration of the nitrogen source in the nutrient solution beinglimited sufficiently to induce said microalgae to produce the singlecell oil at a concentration of at least 2 grams per liter of nutrientsolution; and b) extracting said single cell oil containing at least 20%DHA in the triglyceride form from the biomass.
 2. The pharmaceuticalcomposition according to claim 1 wherein said DHA comprises at least 35%of the weight of the single cell oil.
 3. A dietary supplement comprisinga single cell edible oil containing at least 20% docosahexaenoic acid(DHA) in the triglyceride form, produced by a process comprising:a)cultivating a heterotrophic microalgae of the class Dinophyceae capableof producing said single cell edible oil containing at least 20% DHA inthe triglyceride form, in an aerobic fermentor containing a nutrientsolution having a limiting nitrogen source, said cultivation beingcontinued to achieve a cell density of at least 10 grams of biomass perliter of nutrient solution, the concentration of the nitrogen source inthe nutrient solution being limited sufficiently to induce saidmicroalgae to produce the single cell edible oil at a concentration ofat least 2 grams per liter of nutrient solution; and b) extracting saidsingle cell edible oil containing at least 20% DHA in the triglycerideform from the biomass.
 4. The dietary supplement according to claim 3wherein said DHA comprises at least 35% of the weight of the single celledible oil.
 5. The dietary supplement according to claim 3, wherein saidsupplement comprises a gelatin capsule containing said single celledible oil.
 6. The pharmaceutical composition according to claims 1 or 2wherein the microalgae is Crypthecodinium cohnii.
 7. The dietarysupplement according to claims 3 or 4 wherein the microalgae isCrypthecodinium cohnii.